Ischemia is the state of imbalance of oxygen supply and demand in a part of the body often due to a constriction or an obstruction in the blood vessel supplying that part. The two most common forms of ischemia are cardiovascular and cerebrovascular.
Cardiovascular ischemia is generally a direct consequence of coronary artery disease, and is usually caused by rupture of an atherosclerotic plaque in a coronary artery, leading to formation of thrombus (blood clot), which can occlude or obstruct a coronary artery, thereby depriving the downstream heart muscle of oxygen. Prolonged ischemia can lead to cell death or necrosis, and the region of dead tissue is commonly called an infarct. Patients suffering an event of acute cardiac ischemia often present to a hospital emergency room with chest pain and other symptoms and signs, such as changes to an electrocardiogram (ECG). This type of presentation is referred to as Acute Coronary Syndrome (ACS). A patient diagnosed with ACS requires immediate treatment to avoid irreversible damage to the heart muscle.
Cerebral ischemia is often due to narrowing of the arteries leading to the brain.
Early symptoms of ischemia, which may include headache, dizziness, sensory changes, and temporary loss of certain motor function, are referred to as a Transient Ischemic Attack (TIA). TIAs are a precursor to cerebrovascular accident (CVA or stroke).
The continuum of ischemic disease includes five conditions: (1) elevated blood levels of cholesterol and other lipids; (2) build-up of atherosclerotic plaque and subsequent narrowing of the arteries; (3) reduced blood flow to a body organ (as a result of arterial narrowing or plaque rupture and subsequent thrombus formation); (4) cellular damage to an organ caused by a lack of oxygen; (5) death of organ tissue caused by sustained oxygen deprivation. Stages three through five are collectively referred to as “ischemic disease,” while stages one and two are considered its precursors. It is important to distinguish between the state of ischemia and the disease that leads to it. For example, a patient with coronary artery disease is not always in the state of cardiac ischemia, but a person in the state of cardiac ischemia almost invariably suffers from coronary artery disease.
Together, cardiovascular and cerebrovascular disease accounted for 778,000 deaths in the U.S. in 1998 (2002 Heart and Stroke Statistical Update, 2002 American Heart Association). Additionally, as many as 3 to 4 million Americans suffer from what is referred to as “silent ischemia.” This is a condition where ischemic heart disease is present without the usual and classic symptoms of chest pain or angina.
There is a pressing need for the development and utilization of blood tests able to predict injury to the heart muscle and coronary arteries. Successful treatment of cardiac events depends largely on detecting and reacting to the presence of cardiac ischemia in time to minimize damage. Cardiac enzymes, specifically the creatine kinase isoenzyme (CK-MB) and other markers of cardiac necrosis, specifically myoglobin and the Troponin I and Troponin T biochemical markers, are utilized for diagnosing heart muscle injury. However, these enzymes and markers are only capable of detecting the existence of cell death or necrosis, and therefore have limited or no value in patients who have ischemia without necrosis, such as those in an ischemic state prior to myocardial infarction. Additionally, these enzymes and markers do not show a measurable increase until several hours after the onset of necrosis. For instance, the cardiac troponins do not show a measurable increase above normal in a person's blood test until about four to six hours after the beginning of a heart attack and do not reach peak blood level until about 18 hours after such an event. Thus, the primary shortcoming of using markers of cardiac necrosis for diagnosis of ischemic states is that these markers are only detectable after heart tissue has been irreversibly damaged.
An array of tests are available for diagnosis of cardiac ischemia, particularly in the emergency room (see, for example, Selker, H P et al. (1997) Annals Emergency Medicine 29:13–87). The accepted standard of care is the 12 lead electrocardiogram (ECG or EKG) that, nevertheless, has a clinical sensitivity of less than 50% (see for example, Selker, H P et al. (1997) Annals Emergency Medicine 29:13–87 and Selker, H P et al. Emergency Diagnostic Tests for Cardiac Ischemia, Blackwell Science ISBN 0-632-04304-0 (1997)). Other diagnostic tests include echocardiography, and radionuclide myocardial perfusion imaging.
Diagnosis of coronary artery disease is done either by imaging (e.g., coronary angiography) or by provocative testing, where the intent is to deliberately induce cardiac ischemia and observe the effects. For example, in the ECG exercise stress test, the patient is exercised at an increasing rate to see if symptoms of ischemia are evoked, or if changes indicative of ischemia can be observed on the ECG. Stress ECG is commonly used as an initial screen for coronary artery disease, but is limited by its accuracy rates of only 25–50% (see for example, Froelicher, V F et al. (1988) Ann. Intern. Med. 128(12):965–974). Another commonly used diagnostic test is myocardial perfusion imaging, in which a radioactively tagged chemical is injected during stress testing. Normally metabolizing cardiac tissue is able to take up the radioactively tagged chemical, and is visualized using conventional imaging techniques (PET or SPECT scanning) thereby allowing differentiation between viable and damaged cardiac tissue.
The present invention, however, is believed to be advantageous over the known methods of diagnosis in that it is a simple blood test which will offer comparable accuracy at far lower costs and decreased risk and inconvenience to the patient. It is believed that the present invention provides specificity and sensitivity levels that are comparable in accuracy to current diagnostic standards.
Although there are well established biochemical markers of myocardial necrosis which can be detected in a blood sample using a point of care (POC) instrument, other than as described below in relation to the ACB™ Test, there are no well established biochemical markers for ischemia, and presently no POC instrument for detection of ischemia. An ideal test would be a blood test, preferably administered with a small, simple device providing quick, accurate results that can be used to test for disease, for example at the bedside of a patient with minimal amount of discomfort.
One component of blood is human serum albumin (HSA). Exposure of HSA to ischemic tissue produces modifications to the N-terminus (Bar-Or, D. et al. (2000) J. Emerg. Med. 19:311–315; PCT/US99/22905), and possibly other sites, on the albumin molecule. The N-terminus of albumin has been well characterized as being the primary binding site for several transition metals such as cobalt, nickel and copper (Sadler, P. et al. (1994) Eur. J. Biochem. 220:193–200; Lakusta, H. et al. (1979) J. Inorg. Biochem. 11:303–315; Gasmi, G. et al. (1997) J. Peptide Res. 49:500–509; Predki, P. et al. (1992) Biochem. J. 287:211–215; Lussac, J. et al. (1984) Biochem. 23:2832–38; Matsuoka, J. et al. (1993) J. Biol. Chem. 268:21533–37). Once the N-terminus and possibly other sequestering binding sites have been modified by exposure to ischemic tissue, they are rendered unable to bind metals. This altered albumin is referred to herein as Ischemia Modified Albumin (IMA). Therefore, if a known amount of a transition metal is added to a biological sample (patient sample comprised of whole blood, serum or plasma, urine, cerebrospinal fluid, saliva and the like), normal albumin and IMA can be differentiated by monitoring the amount of non-binding metal. Metal added to the sample will be sequestered at the N-terminus and possibly other sites on albumin more frequently in a non-ischemic sample than in an ischemic sample in which albumin has been modified in such a way that it can no longer bind the metal. The metal not sequestered at the N-terminus and possibly other sites on albumin in the samples can then be detected and quantified using the Albumin Cobalt Binding (ACB) Test (Ischemia Technologies, Inc., Denver, Colo.), which, as described in PCT/US99/22905, filed Oct. 1, 1999, and U.S. Pat. No. 5,227,307, uses calorimetric methods to determine the amount of IMA present in the sample. PCT/US99/22905 also provides a detailed description of the N-terminal modifications to albumin during an ischemic event. Studies have been conducted demonstrating the clinical utility of IMA via the ACB test in diagnosing and risk stratifying patients.
The ACB Test uses a laboratory chemistry analyzer to quantify IMA, but presently, there are no POC tests for ischemia. An ideal test would be a blood test, preferably administered with a small, simple device providing quick, accurate results referenced to a standard curve with a quality control system that can be used to test for disease at the bedside of a patient with minimal amount of discomfort. The aim of this invention is to provide such a diagnostic test.
It is an object of the subject invention to provide a diagnostic test that detects a change in a biological molecule by detecting a signal produced or altered by the change in the biological molecule, wherein the change relates to the binding of a metal to a portion of the biological molecule.
Another object is to provide a diagnostic test that determines a difference in current or potential measurements in biological fluids from ischemic patients and non-ischemic individuals, wherein the samples are first combined with cobalt or another transition metal.
It is another object of the subject invention to provide an electrochemical assay for detecting a biological condition via detection of metal binding with a biological sample, wherein there is a difference in signal relative to the amount of additives such as metal, complexing reagent or other reagents added to the biological sample where the signal is standardized using a calibration and quality control system.
Another object of the subject invention is to use data processing techniques to identify features of the electrochemical output data from an electrochemical assay for determining the differences between ischemic and non-ischemic individuals.
It is a further object of the subject invention to provide an apparatus for assaying a patient's condition at the patient's bedside.